The basic unit of a biological system is a single cell, and malfunctions at the single-cell level can result in devastating diseases, such as in cancer metastasis, where a single cell seeds the formation of a distant tumor. Given that a single cell contains a diverse set of molecules at low copy numbers, high-resolution and high-sensitivity techniques in microscale chemical separation play an important role in single-cell analysis. For many single-cell studies, it is insufficient simply to detect a series of separated peaks using fluorescence. Because of the complexity of the cellular contents, the detected peaks likely need to be further separated (e.g., a second-dimensional analysis) or analyzed with complementary high-sensitivity techniques.
In microscale separation, the goal is to separate individual analyte species of a complex mixture into distinct bands. After the detection of each band, however, the integrity of the separated components cannot be easily preserved for additional analysis or manipulation, owing to molecular diffusion. This challenge is especially acute in high-resolution liquid separation techniques, such as in capillary electrophoresis (CE) and microscale high-performance liquid chromatography (HPLC), because of the extremely small volumes and narrow bands involved and because molecular diffusion scales quadratically with the inverse of distance.
In CE, for example, sample volumes are often in the nanoliter range or smaller, and where the number of theoretical plates often range up to the millions. In some chip-based systems, or when very small-bore capillaries are used, sample volumes can range down to the femtoliter regime. In such systems, it is extremely difficult to maintain the contents of the separated peaks after their detection.
Attempts have been made to address the issue of material compartmentalization after separation. For example, elastomeric valves and sub-nanoliter chambers have been used to capture separated CE bands for single-molecule studies. In the context of two-dimensional (2D) CE separation, methods have been developed for interfacing two fused-silica CE capillaries such that the separated CE bands can be transferred from one capillary onto another for 2D separation and analysis.
Despite recent advances, a technique that allows for the preservation of chromatographically-separated liquid materials, such that further analysis of the materials can be performed while maintaining the integrity of the original separation, would allow for further improvements in liquid chromatography and related analytical techniques.
Relatedly, on-line sampling is required to monitor the progress of many chemical and biological processes, such as in chemical synthesis and bioreactors. In real-time sampling, however, the withdrawn samples are dispersed throughout the sampling tubing as a result of axial dispersion generated by the parabolic profile of the pressure-driven flow and because of the effects of diffusion. The methods currently employed to test such real-time reaction profiles have not provided a sufficient solution to the issues of dispersion and the inaccuracies in the resulting analyses that result from such dispersion.
In addition to sample dilution due to dispersion and diffusion effects, sample integrity in on-line sampling can be further compromised by contaminates deposited on the channel wall of the device during prior sample testing.